Isolated rat liver mitochondria are being studied to determine the physicochemical properties of the internal aqueous phase. This matrix phase is normally assumed to have properties of bulk aqueous solutions; however previous studies with nonelectrolytes have clearly shown this assumption to be incorrect. This finding has important consequences for the calculation of protonmotive force, delta pH and membrane potential from ion distributions, in which activity coefficient ratios are normally assumed to be 1.0. A heterogenous two-phase model, in which one phase is "bulk" and the other is "abnormal" in its solution properties has been found to be consistent with non-electrolyte distribution data. Organic acid distributions will be studied using radioisotopes of acetate, malate, phosphate and other anions. Delta pH will be calculated on the basis of both models. It is necessarry to control cation transport and content during these studies. We have developed, and will extend, procedures for stopping the mitochondrial K/H carrier and for preparing tetraethylammonium substituted mitochondria. K ion kinetics will be studies with ion electrodes and atomic absorption spectroscopy.